Photoreceptor outer segment membranes, the site of initial photon capture initiating phototransduction, undergo renewal with total replacement occurring at ten day intervals throughout life. A challenge to cellular integrity concerns the post biosynthetic delivery of replacement proteins from the inner, to the outer, segment over the lifetime of the photoreceptor. Complex trafficking pathways require multiple components, such as acyl-binding proteins, Rab GTPases and centrins. This application will identify: i) key Rab GTPases involved in vesicular transport of membrane protein, and ii) centrins participating in ciliary transport. Aim 1 will focus on Rab GTPases in photoreceptor vesicular transport. Rab (Ras analog in brain) proteins are members of the Ras supergene family involved mainly in membrane protein trafficking. More than 70 distinct Rab polypeptides have been identified. Aim 1a focuses on Rab8 and Rab11 isoforms which are known to be important for photoreceptor trafficking in Xenopus and Drosophila models. Surprisingly, a mouse Rab8a/Rab11a double knockout does not produce a defective trafficking phenotype. Therefore, we will focus on closely-related isoforms to identify key Rab proteins responsible for membrane protein organization and rhodopsin trafficking. Aim 1b investigates Rab28, shown recently associated with recessive human cone/rod dystrophy. Aim 2 will examine the function of centrosomal proteins, called centrins, which are 20kD EF- hand calcium binding proteins (four EF-hand motifs) of the calmodulin superfamily. Centrins were first described in unicellular green algae where they are associated with the flagellar basal apparatus. Photoreceptor centrins (isoforms 1-4) are located in the ciliary lumen and basal body where they associate with transducin via its T?-subunits. One hypothesis is that centrins may be involved in regulation of transducin translocation from the outer, to the inner, segment or vice-versa. As preliminary results, we found that centrin1 is required for the nucleus-basal body connection during mouse spermiogenesis, and centrin2 regulates trafficking of ACIII and channel subunits in olfactory sensory neurons. This aim seeks to identify the roles of photoreceptor centrin3 and centrin4 which associate strongly with the basal body and/or connecting cilium axoneme. We expect to generate mouse models of syndromic disease with retina degeneration, e.g., Joubert or Senior-Loken syndromes.